1. Field of Invention
The invention relates to nucleic acid multiplexes, and more particularly to sensitive methods for accurately assaying triplex and quadruplex nucleic acid complexes employing catalytic hybridization.
2. Description of Related Art
Although nucleic acid duplexes are the most widely studied type of multiple-strand nucleic acid structures, it has been discovered that nucleic acids also form triplex and quadruplex structures under certain conditions.
Until recently, hybridization among three nucleic acid strands to form a triplex was widely believed to be confined to very limited species of nucleic acids (e.g., polypurine or polypyrimidine sequences). See, e.g., Floris et al., “Effect of cations on purine-purine-pyrimidine triple helix formation in mixed-valence salt solutions,” 260 Eur. J. Biochem. 801-809 (1999). Moreover, triplex formation or hybridization was thought to be based on Hoogsteen binding between limited varieties of adjacent nucleobases, rather than Watson-Crick base pairing. See, e.g., Floris et al. and U.S. Pat. No. 5,874,555 to Dervan et al. However, the inventors have recently disclosed in several patent applications that triplex nucleic acids based on Watson-Crick base pairing can be created and used as the basis for a highly accurate and sensitive assay for specific binding. See U.S. patent applications Ser. Nos. 09/613,263 and 09/468,679, respectively filed Jul. 10, 2000 and Dec. 21, 1999.
As was the case with triplex nucleic acids, the conventional wisdom regarding quadruplex nucleic acids has been that such peculiar structures only exist under relatively extreme conditions for a relatively narrow class of nucleic acids. In particular, Sen et al. (Nature 334:364-366 (1988)) disclosed that guanine-rich oligonucleotides can spontaneously self-assemble into four-stranded helices in vitro. Sen et al. (Biochemistry 31:65-70 (1992)) disclosed that these four-stranded complexes can further associate into superstructures composed of 8, 12, or 16 oligomers.
Marsh et al. (Biochemistry 33:10718-10724 (1994), and Nucleic Acids Research 23:696-700 (1995)) disclosed that some guanine-rich oligonucleotides can also assemble in an offset, parallel alignment, forming long “G-wires”. These higher-order structures are stabilized by G-quartets that consist of four guanosine residues arranged in a plane and held together through Hoogsteen base pairings. According to Sen et al. (Biochemistry 31:65-70 (1992)), at least three contiguous guanines within the oligomer are critical for the formation of these higher order structures.
It has been suggested that four-stranded DNAs play a role in a variety of biological processes, such as inhibition of HIV-1 integrase (Mazumder et al., Biochemistry 35:13762-13771 (1996)), formation of synapsis during meiosis (Sen et al., Nature 334:364-366 (1988)), and telomere maintenance (Williamson et al., Cell 59:871-880 (1989)); Baran et al., Nucleic Acids Research 25:297-303 (1997)).
It has been further suggested that controlling the production of guanine-rich quadruplexes might be the key to controlling such biological processes. For example, U.S. Pat. No. 6,017,709 to Hardin et al. suggests that telomerase activity might be controlled through drugs that inhibit the formation of guanine quartets.
U.S. Patent No. 5,888,739 to Pitner et al. discloses that G-quartet based quadruplexes can be employed in an assay for detecting nucleic acids. Upon hybridization to a complementary oligonucleotide, the G-quartet structure unfolds or linearizes, thereby increasing the distance between a donor and an acceptor on different parts of the G-quartet structure, resulting in a decrease in their interaction and a detectable change in a signal (e.g., fluorescence) emitted from the structure.
U.S. Pat. No. 5,912,332 to Agrawal et al. discloses a method for the purification of synthetic oligonucleotides, wherein the synthetic oligonucleotides hybridize specifically with a desired, full-length oligonucleotide and concomitantly form a multimer aggregate, such as quadruplex DNA. The multimer aggregate containing the oligonucleotide to be purified is then isolated using size-exclusion techniques.
Conventional assays for nucleic acids have generally been based on a duplex hybridization model, wherein a single-stranded probe specifically binds to a complementary single-stranded target sequence.
Peter Duck and his colleagues have disclosed particularly sensitive methods for detecting duplex hybridization, based on catalytic hybridization technology. See, e.g., U.S. Pat. Nos. 4,876,187, 5,011,769, 5,660,988 and 5,731,146, all to Duck et al. Briefly stated, catalytic hybridization is a method in which a large stoichiometric excess of probe is added to target in the presence of a cleaving agent adapted to specifically cleave duplexed probe. The cleaved fragments of the probe then dehybridize to provide detectable probe fragments distinguishable from intact probes, and a recycled target available for hybridization with additional intact probes. Thus, the target acts as a sort of catalyst for the cleaving step; hence the name “catalytic hybridization” (a term apparently coined by Walder et al. in U.S. Pat. No. 5,403,711).
As a single target can catalyze the cleavage of a virtually unlimited number of intact probes to provide a multitude of detectable probe fragments, the signal is amplified relative to more conventional detection methods in which each target accounts for only a single signal once hybridized with a probe.
Despite the foregoing developments, the full potential of catalytic hybridization has neither been fully appreciated nor fully exploited.
All references cited herein are incorporated herein by reference in their entireties.